Carboxylic acids and derivatives thereof are widely used as specialty chemicals for applications in polymers, foods, pharmaceuticals, and cosmetics. Succinic acid, for example, is useful for the production of such plastic precursors as 1,4-butanediol (BDO), tetrahydrofuran, and gammabutyrolactone. New products derived from succinic acid are under continual development, including the development of polyester. Polyester is made by linking succinic acid and BDO. Generally, esters of succinic acids have the potential of being new, "green" solvents that can supplant more harmful solvents and serve as precursors for millions of pounds of chemicals annually at a total market value of over one billion dollars.
The production of carboxylic acids, such as malic acid, succinic acid and fumaric acid, from renewable feedstocks (in this case through fermentation processes) is an avenue to supplant the more energy intensive methods of deriving such acids from nonrenewable sources. Succinate is an intermediate for anaerobic fermentations by propionate-producing bacteria but those processes result in low yields and concentrations.
Many succinic acid-producing organisms have been isolated, such as the anaerobic rumen bacteria, Bacteroides ruminicola and Bacteroides amylophilus. However, rumen organisms are characteristically unstable in fermentation processes. Another succinic acid-producing organism is Anaerobiospirillum succiniciproducens (A. succiniciproducens). Several patents have been issued on the use of this organism to produce succinic acid in an anaerobic fermentation process. One such patent by Glassner et al, U.S. Pat. No. 5,143,834, outlines the use of this organism in fermentation processes to naturally produce succinic acid in moderate yields. However, fermentation processes using A. succiniciproducens have a number of problems. One problem is that the organism is a strict anaerobe, its cultivation must be performed in an environment absolutely free of oxygen. The propagation of this organism in a commercial fermentation plant is difficult and requires highly skilled workers. A. succiniciproducens is also difficult to handle even in laboratory-scale practice and tends to degenerate under unfavorable conditions. Its degeneracy cannot be reversed. The organism has never been used in a commercial fermentation process. In other words, production-scale fermentation experience with this particular organism is non-existent. Furthermore, the organism requires an external supply of carbon dioxide to achieve a high yield of succinic acid. In a fermentation process, a stream of pure carbon dioxide must be sparged into the fermentation broth. A. succiniciproducens produces a mixture of succinic and acetic acids at a succinate:acetate molar ratio of about 2. The presence of acetic acid at high concentrations in the fermentation broth increases the cost of succinic acid purification. Production of the acetate co-product illustrates that one-third of the expensive glucose is not converted to succinate. Furthermore, the A. succiniciproducens host strain has been shown to be not highly osmotolerant in that it does not tolerate high concentrations of salts and is further inhibited by moderate concentrations of product. Another problem that the use of A. succiniciproducens presents is that medium preparation for the inoculum requires the addition of tryptophan and also requires the mixing of four different solutions, one of which contains corrosive and toxic H.sub.2 S.
It has long been known that a mixture of acids are produced from E. coli fermentation, as elaborated by J. L. Stokes in 1949 "Fermentation of glucose by suspensions of Escherichia coli," J. Bacteriol. 57:147-158. However, for each mole of glucose fermented, only 1.2 moles of formic acid, 0.1-0.2 moles of lactic acid, and 0.3-0.4 moles of succinic acid are produced. As such, efforts to produce carboxylic acids fermentatively have resulted in relatively large amounts of growth substrates, such as glucose, not being converted to desired product.
Fairoz Mat-Jan et al describes in the J. Bacteriol., v. 171 (1989), pp. 342-348, a study conducted on mutants of Escherichia coli deficient in the fermentative AND-linked lactate dehydrogenase (ldh) that had been isolated showed no growth defects under anaerobic conditions unless present together with a defect in pyruvate formate lyase (pfl). Double mutants (pfl ldh) were unable to grow anaerobically on glucose or other sugars even when supplemented with acetate, whereas pfl mutants can do so. The study did not discuss nor did it investigate the production of succinic acid or dicarboxylic acids.
Although the succinate ion is a common intermediate in the metabolic pathway of several anaerobic microorganisms, a need exists in the art for a fermentation process to economically produce succinic acid as well as other carboxylic acids such as malic acid and fumaric acid, in large amounts or with high yields. The process should utilize low cost nutrients and substrates, the rate of fermentation should be high for high productivity, and the product concentration in the fermentation broth should be high.